Networks of Splice Factor Regulation by Unproductive Splicing Coupled With NMD Abstract Virtually all multi-exon genes undergo alternative splicing (AS) to generate multiple protein isoforms (Wang et al., 2008). Alternative splicing is regulated by splicing factors (SF), such as the serine/arginine rich (SR) protein famil (Long and Caceres, 2009). All SR factors have alternative isoforms that contain in-frame premature termination codons (PTCs) that are recognized by the nonsense mediated mRNA decay (NMD) pathway and degraded (Ni et al., 2007; Lareau et al., 2007; Ishigaki et al., 2001). Generally, NMD is thought to play a protective role, by destroying truncated transcripts, but it also regulates gene expression through alternative splicing coupled with NMD. It has been shown that several SFs, including many SR proteins, affect splicing of their own pre-mRNA (Sureau et al., 2001; Sun et al., 2010). In this mode of negative feedback loop regulation, high levels of the SF cause its own pre-mRNA to be spliced into an unproductive isoform and degraded, resulting in lower protein levels (Sureau et al., 2001a). For example, SR proteins SRSF1, SRSR2, SRSF3, and SRSF7 are known to regulate their own expression by coupling alternative splicing and NMD (Sun et al., 2010; Sureau et al., 2001; nk et al., 2012b), also hnRNP L (Rossbach et al., 2009) and PTB (Spellman et al., 2005) are regulated in the same manner. There is anecdotal evidence that this mode of regulated unproductive splicing and translation (Green et al., 2003) is used in an inter-factor fashion, creating a splicing factor regulatory network (SF network). We hypothesize that the splicing regulation extends to all SR and other SFs. To date, there has been no systematic study of human splicing factors' gene regulation via NMD. A literature search allowed us to construct an SF network. This network is rich in protein-mRNA interactions, providing an intriguing hint that many SFs undergo alternative splicing coupled with NMD. Therefore we propose to conduct a comprehensive experimental survey to identify SFs that are regulated by alternative splicing coupled with NMD, to determine the effect of SF perturbation on other splicing factors, and to gain a functional insight on this regulation by looking at direct protein-mRNA binding sites. To identify SFs that are regulated by NMD, we will inhibit the NMD pathway in human cell lines via RNAi against essential NMD factors (Gatfield et al., 2003). Transcripts that undergo NMD will be up regulated in the NMD-inhibited environment and will be identified through the RNA-seq analysis. To determine the effect of SF perturbation on other splicing factors, we will overexpress and knockdown individual factors and will observe transcriptome perturbations using RNA-seq experiments and quantitative reverse transcriptase PCR (qRT-PCR) assay using splicing factors specific primers. To gain a functional insight on NMD regulation, we will look at direct protein-mRNA binding sites through UV cross-linking and immunoprecipitation (iCLIP) experiments of individual SFs followed by genome wide RNA-seq analysis with careful filtering for secondary effects. This experimental approach will allow us to determine the network of splicing factor regulation via alternative splicing coupled with NMD.